Elimination of primer-primer interactions during primer extension

Abstract

The invention comprises a method of amplifying nucleic acids by primer extension with reduced formation of primer-primer byproducts.

The invention claimed is: 
     
       1. A method of amplifying a target nucleic acid in a sample with reduced primer-primer interaction comprising:
 (a) a primer extension step, wherein the sample is contacted with: (i) a nucleic acid polymerase, and (ii) a first primer which is a single-stranded oligonucleotide comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase, the first primer comprising two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence, such that a gap is created between the ends of the two arms when the first primer hybridizes to the target nucleic acid, wherein the polymerase fills the gap between the ends of the two arms; and 
 (b) an exponential amplification step wherein the sample is contacted with a second primer complementary to the primer extension product and a polymerase tolerant of the modified nucleotide. 
 
     
     
       2. The method of  claim 1 , wherein the first primer comprises binding sites for universal amplification primers and the second primer is a universal primer. 
     
     
       3. The method of  claim 1 , wherein the modified nucleotide is uracil. 
     
     
       4. A reaction mixture for synthesizing nucleic acid strands according to  claim 1 , comprising a target nucleic acid, a nucleic acid polymerase and a first primer which is a single-stranded oligonucleotide consisting of two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence and comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase. 
     
     
       5. The method of  claim 1 , further comprising a step of joining the ends of the extended first primer by ligation. 
     
     
       6. The method of  claim 5 , further comprising prior to step (b), contacting the sample with an endonuclease. 
     
     
       7. The method of  claim 1 , wherein the first primer comprises a unique molecular barcode. 
     
     
       8. The method of  claim 1 , wherein the second primer comprises a sample barcode. 
     
     
       9. The method of  claim 1 , further comprising sequencing the amplified nucleic acid from step (b). 
     
     
       10. The method of  claim 9 , wherein a universal sequencing primer site is located in the second primer.